Bottom Line:
Administration of 5-fluorouracil to mice induces myelosuppression, but QuOPs survive and differentiate into osteoclasts in response to an active vitamin D(3) analogue given to those mice.Mononuclear cells expressing c-Fms and RANK but not Ki67 are detected along bone surfaces in the vicinity of osteoblasts in RANKL-deficient mice.These results suggest that QuOPs preexist at the site of osteoclastogenesis and that osteoblasts are important for maintenance of QuOPs.

ABSTRACTOsteoclasts are multinucleated cells that resorb bone. Although osteoclasts originate from the monocyte/macrophage lineage, osteoclast precursors are not well characterized in vivo. The relationship between proliferation and differentiation of osteoclast precursors is examined in this study using murine macrophage cultures treated with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB (RANK) ligand (RANKL). Cell cycle-arrested quiescent osteoclast precursors (QuOPs) were identified as the committed osteoclast precursors in vitro. In vivo experiments show that QuOPs survive for several weeks and differentiate into osteoclasts in response to M-CSF and RANKL. Administration of 5-fluorouracil to mice induces myelosuppression, but QuOPs survive and differentiate into osteoclasts in response to an active vitamin D(3) analogue given to those mice. Mononuclear cells expressing c-Fms and RANK but not Ki67 are detected along bone surfaces in the vicinity of osteoblasts in RANKL-deficient mice. These results suggest that QuOPs preexist at the site of osteoclastogenesis and that osteoblasts are important for maintenance of QuOPs.

fig3: Incorporation of BrdU into nuclei of osteoclasts in vivo. (A) BrdU incorporation into nuclei in osteoclasts at different growth stages of mice. Pregnant mice at E13.5 and 3- and 7-wk-old mice were given 1 mg/ml BrdU in drinking water for 1 wk. Newborn, 4-, and 8-wk-old mice administered with BrdU for 1 wk were killed, and tibiae were recovered. Sections of tibiae were prepared and double stained for TRAP (red) and BrdU (brown). Arrows indicate the BrdU+ nuclei in osteoclasts (right). BrdU+ and BrdU− nuclei in osteoclasts were counted, and percentages of BrdU+ nuclei in osteoclasts were determined (left). Changes in the body weight of mice are shown. (B) BrdU incorporation into nuclei in osteoclasts in adult mice. 7-wk-old mice were given 1 mg/ml BrdU in drinking water for 7 wk. Tibiae were recovered and subjected to TRAP and BrdU staining. Arrows indicate BrdU+ nuclei in osteoclasts (right). Percentages of BrdU+ nuclei in osteoclasts were determined (left). Results are expressed as the mean ± SD for three animals.

Mentions:
We next examined in vivo labeling of nuclei of osteoclasts with BrdU. BrdU in 1 mg/ml drinking water was administered for 1 wk to mice at different developmental stages, and the incorporation of BrdU into the nuclei of osteoclasts was evaluated in tibiae (Fig. 3). When pregnant mice at 13.5 d postcoitum (embryonic day 13.5 [E13.5]) were given BrdU in drinking water for 1 wk, ∼50% of the nuclei of osteoclasts in newborn mice were labeled with BrdU. Similarly, 3- and 7-wk-old mice were given BrdU in drinking water for 1 wk. About 30% of the nuclei of osteoclasts were labeled with BrdU in 4-wk-old mice. In contrast, most nuclei of osteoclasts were BrdU negative (BrdU−) in 8-wk-old mice. Thus, the BrdU labeling of osteoclasts was inversely correlated with the growth of mice (Fig. 3 A). We then examined the lifespan of QuOPs in adult mice. When 7-wk-old mice were given BrdU for an additional 7 wk, only 50% of the nuclei of osteoclasts were labeled with BrdU (Fig. 3 B). Most of the bone marrow cells around osteoclasts were BrdU positive (BrdU+) in mice treated with BrdU for 7 wk. Previous studies have shown that the lifespan of osteoclasts is 2–4 wk in humans and mice (Kodama et al., 1993; Riggs and Parfitt, 2005). These results suggest that the lifespan of QuOPs is at least 4 wk longer.

fig3: Incorporation of BrdU into nuclei of osteoclasts in vivo. (A) BrdU incorporation into nuclei in osteoclasts at different growth stages of mice. Pregnant mice at E13.5 and 3- and 7-wk-old mice were given 1 mg/ml BrdU in drinking water for 1 wk. Newborn, 4-, and 8-wk-old mice administered with BrdU for 1 wk were killed, and tibiae were recovered. Sections of tibiae were prepared and double stained for TRAP (red) and BrdU (brown). Arrows indicate the BrdU+ nuclei in osteoclasts (right). BrdU+ and BrdU− nuclei in osteoclasts were counted, and percentages of BrdU+ nuclei in osteoclasts were determined (left). Changes in the body weight of mice are shown. (B) BrdU incorporation into nuclei in osteoclasts in adult mice. 7-wk-old mice were given 1 mg/ml BrdU in drinking water for 7 wk. Tibiae were recovered and subjected to TRAP and BrdU staining. Arrows indicate BrdU+ nuclei in osteoclasts (right). Percentages of BrdU+ nuclei in osteoclasts were determined (left). Results are expressed as the mean ± SD for three animals.

Mentions:
We next examined in vivo labeling of nuclei of osteoclasts with BrdU. BrdU in 1 mg/ml drinking water was administered for 1 wk to mice at different developmental stages, and the incorporation of BrdU into the nuclei of osteoclasts was evaluated in tibiae (Fig. 3). When pregnant mice at 13.5 d postcoitum (embryonic day 13.5 [E13.5]) were given BrdU in drinking water for 1 wk, ∼50% of the nuclei of osteoclasts in newborn mice were labeled with BrdU. Similarly, 3- and 7-wk-old mice were given BrdU in drinking water for 1 wk. About 30% of the nuclei of osteoclasts were labeled with BrdU in 4-wk-old mice. In contrast, most nuclei of osteoclasts were BrdU negative (BrdU−) in 8-wk-old mice. Thus, the BrdU labeling of osteoclasts was inversely correlated with the growth of mice (Fig. 3 A). We then examined the lifespan of QuOPs in adult mice. When 7-wk-old mice were given BrdU for an additional 7 wk, only 50% of the nuclei of osteoclasts were labeled with BrdU (Fig. 3 B). Most of the bone marrow cells around osteoclasts were BrdU positive (BrdU+) in mice treated with BrdU for 7 wk. Previous studies have shown that the lifespan of osteoclasts is 2–4 wk in humans and mice (Kodama et al., 1993; Riggs and Parfitt, 2005). These results suggest that the lifespan of QuOPs is at least 4 wk longer.

Bottom Line:
Administration of 5-fluorouracil to mice induces myelosuppression, but QuOPs survive and differentiate into osteoclasts in response to an active vitamin D(3) analogue given to those mice.Mononuclear cells expressing c-Fms and RANK but not Ki67 are detected along bone surfaces in the vicinity of osteoblasts in RANKL-deficient mice.These results suggest that QuOPs preexist at the site of osteoclastogenesis and that osteoblasts are important for maintenance of QuOPs.

ABSTRACTOsteoclasts are multinucleated cells that resorb bone. Although osteoclasts originate from the monocyte/macrophage lineage, osteoclast precursors are not well characterized in vivo. The relationship between proliferation and differentiation of osteoclast precursors is examined in this study using murine macrophage cultures treated with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB (RANK) ligand (RANKL). Cell cycle-arrested quiescent osteoclast precursors (QuOPs) were identified as the committed osteoclast precursors in vitro. In vivo experiments show that QuOPs survive for several weeks and differentiate into osteoclasts in response to M-CSF and RANKL. Administration of 5-fluorouracil to mice induces myelosuppression, but QuOPs survive and differentiate into osteoclasts in response to an active vitamin D(3) analogue given to those mice. Mononuclear cells expressing c-Fms and RANK but not Ki67 are detected along bone surfaces in the vicinity of osteoblasts in RANKL-deficient mice. These results suggest that QuOPs preexist at the site of osteoclastogenesis and that osteoblasts are important for maintenance of QuOPs.